Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.

Gamma-irradiation stimulates homology-directed DNA double-strand break repair in Drosophila embryo.

To test the DNA double-strand break (DSB) repair activities present in Drosophila early embryos, we have analyzed the circularization of a microinjected linear plasmid. In order to study repair by homologous recombination, the linear plasmid was injected with an homologous fragment encompassing the break. After extraction from embryos, repair products were analyzed directly by PCR and after their cloning into bacteria. We demonstrate, in addition to the repair by homologous recombination, the presence of an efficient end-joining activity in embryos. Plasmid circularization by end-joining was accompanied by short deletions frequently associated with non-random insertions. Most importantly, pre-irradiation of embryos specifically enhanced the accurate repair by homologous recombination. Such a stimulation is described for the first time in the context of a whole higher organism.

Evidence for a multistep control in transposition of I factor in Drosophila melanogaster.

Drosophila melanogaster strains belong to one of two interactive categories, inducer (I) or reactive (R), with respect to the I-R system of hybrid dysgenesis. The dysgenic interaction results from the presence of several transposition-competent copies of a LINE-like element, the I factor, only in the genome of I strains. When a cross is performed between I males and R females, I factor transposes at high frequency in the germ line of F1 daughters, known as SF females. This transposition burst results in the sterility of SF females. I factor transposes by reverse transcription of a full-length transcript. Specific RT-PCR experiments were done to compare the amount of I factor transcript in samples corresponding to various transposition frequencies. The sensitivity of the method allowed the ready detection of the I factor RNA in every tissue and genetic background examined. Comparison of amplification signals suggests that I factor activity in ovaries is regulated at different levels. First, the amount of I factor RNA subjected to negative and positive regulation. Whereas the negative control, which limits transposition in nonpermissive contexts, may be exerted by an I factor encoded repressor function, the positive control is linked to reactivity level, a cellular state maternally inherited from R mothers. Additionally, negative regulation is also exerted downstream of I factor RNA. This differs notably from previous conclusions in which transcription was envisaged as the main level of regulation of the I factor transposition.

Episomal amplification or chromosomal integration of the viral genome: alternative pathways in hamster polyomavirus-induced lymphomas.

The state and expression of the hamster polyomavirus genome in a large panel of virus-induced lymphomas have been investigated. The viral genome is present within tumor cells either as abundant nonrandomly deleted extrachromosomal copies or as a single copy integrated into cellular DNA. We show that these two physical states are likely to be functionally equivalent: first, deletion and integration of the viral genome both inactivate the late coding region; second, the amount of viral early RNAs yielded by a single integrated copy appears to be very similar to that associated with several thousands of extrachromosomal copies of the viral genome. These data underline two essential requisites for hamster polyomavirus to become lymphomagenous: suppression of the late coding functions of the viral genome and expression of the viral oncogenes above a threshold level.

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.

Viral genomes maintained extrachromosomally in hamster polyomavirus-induced lymphomas display a cell-specific replication in vitro.

Hamster polyomavirus causes lymphomas when injected into newborn Syrian hamsters. Large amounts of extrachromosomal viral genomes are accumulated in the lymphoma cells. These genomes are characterized by deletions affecting the late coding region as well as a specific part of the noncoding regulatory region. By contrast with wild-type genomes, lymphoma-associated genomes replicate in a lymphoblastoid cell line but not in a fibroblastic cell line. The deletion acts in a cis-dominant manner and is the primary determinant of this host-range effect on replication. The boundaries of the regulatory region necessary for viral DNA replication in the two cell contexts have been defined. The regulatory region can be functionally divided in two domains: one domain (distal from the origin of replication) is necessary for viral genome replication in fibroblasts, whereas the other domain (proximal to the origin of replication) is functional only in the lymphoblastoid cell context and contains the sequence specifically conserved in the lymphoma-associated genomes. This sequence harbors a motif recognized by a lymphoblastoid cell-specific trans-acting factor.

Mutations within the hamster polyomavirus large T antigen domain involved in pRb binding impair virus productive cycle and immortalization capacity.

Hamster polyomavirus (HaPV) causes lymphoma and leukemia when injected into newborn Syrian hamsters and achieves full transformation of rodent fibroblasts in vitro. It offers a comprehensive model to study at a molecular level the contributions of the viral oncogenes to neoplastic transformation in vitro and in the animal. We have investigated the ability of HaPV large T antigen to form a complex with the product of the retinoblastoma gene (pRb) in vitro. In this report, we demonstrate that HaPV large T antigen can indeed complex the pRb polypeptide. In order to investigate to what extent this interaction might contribute to tumor induction by the virus, we have introduced two different point mutations within the putative pRb-binding sequence of large T antigen, and as a preliminary to in vivo experiments we have studied their effects in vitro on some biological activities relevant to tumor induction. We show that the substitution (Glu-134-->Lys) obliterates pRb binding, suggesting that Glu-134 participates in the interaction between pRb and large T antigen, whereas the substitution (Glu-135-->Lys) has no effect. The Lys-134 mutation is strongly deleterious to the immortalization capacity of the viral genome, whereas the Lys-135 mutation has no effect. Neither of the two mutations affects the capacity of the viral genome to induce foci formation in the rat established cell line F111. These results indicate that the interaction between large T and pRb is required in the immortalization process but irrelevant to transformation. Both mutants show at least partial impairment of replication and productive cycle.

Distinct segments of the hamster polyomavirus regulatory region have differential effects on DNA replication.

The replication of plasmids containing various fragments of the hamster polyomavirus (HaPV) DNA non-coding region was tested in a permissive hamster cell line. We first investigated the importance of some methodological parameters including the time course and the amount of transfecting plasmid DNA and have shown that these factors can greatly influence the relative amount of newly replicated DNA accumulated within the transfected cells. Taking these into account, quantitative comparisons could be made showing the effect of various parts of the regulatory sequence on the HaPV DNA replication.

Early gene expression in lymphoma-associated hamster polyomavirus viral genomes.

Hamster polyomavirus (HaPV) is the causal agent of hair follicle epithelioma in hamsters belonging to a colony bred in Berlin-Buch. These tumors shed virus particles that are assembled in the keratinized layer of the epidermis. By contrast, HaPV induces lymphomas after inoculation into newborn hamsters from a distinct colony bred in Potsdam. These lymphoid tumors accumulate massive amounts of episomal viral genomes characterized by deletions that alter specifically the regulatory and the late coding sequences. Assuming that these alterations of the regulatory region may affect the transcription of the viral oncogenes in the tumor cells, the transcriptional activity of the wild-type and deleted early promoters have been studied in vitro in transient chloramphenicol acetyltransferase (CAT) expression assays. These assays performed in various cell types demonstrate that both versions of the HaPV early promoter carry a weak constitutive activity. Simultaneous expression of the HaPV early gene products leads to a strong stimulation of CAT activity with a concomitant activation of the replication of the plasmid constructs. The results obtained with origin-defective CAT vectors indicate that the replication contributes significantly to the stimulating effect of the early gene products. Indeed, transfection of massive amounts of CAT vectors that are unable to replicate can simulate the dosage effect of replication and also leads to measurable CAT activities. Under these conditions, the wild-type promoter is more active than the deleted version, indicating that sequences within the deletion carry a distinct stimulatory effect on transcription. This conclusion is supported by the observation that the lymphoma cells contain a low level of early transcripts, indicating that the deleted episomal viral templates accumulated in these tumors carry a weak transcriptional activity.

[Oncogenes as molecular basis of carcinogenesis]. / Les oncogènes comme base moléculaire de la cancérogenèse.

Oncogenes are cellular genes altered by different mechanisms in numerous human tumors. Under this modified form, because of their intervention in the cellular division and differentiation phenomenons, they can play a role in the malignant process. Nevertheless, there is no univocal link between an oncogene alteration and one special type of tumour. Moreover, the role of other biological parameters, which can influence the oncogenes action, must be considered to explain the generation of a cancer.

Analysis of the hamster polyomavirus infection in vitro: host-restricted productive cycle.

In a search for a host fully permissive for the hamster polyomavirus (HaPV) productive cycle in cell culture, the replication of the viral genome has been assayed in a panel of murine and hamster cell types. These experiments led to the conclusion that hamster cells represent the most permissive host for HaPV DNA replication although some murine cells also permit replication of the viral DNA. A single burst of infectious particles is demonstrable in some replication-competent cells, but the outcome of the infection appears to be clearly host dependent. In one hamster cell line (GD36), the virus can be propagated by successive productive cycles. In other hamster cells, despite a successful initial virus burst following transfection of viral DNA, a block in the production of virus particles seems to prevent the spread of infection. This type of restriction level may play a role in the in vivo host range of HaPV.